Nicolas Severe

Ischemic Stroke Activates Hematopoietic Bone Marrow Stem Cells

Rationale: The mechanisms leading to an expanded neutrophil and monocyte supply after stroke are incompletely understood. Objective: To test the hypothesis that transient middle cerebral artery occlusion (tMCAO) in mice leads to activation of hematopoietic bone marrow stem cells. Methods and Results: Serial in vivo bioluminescence reporter gene imaging in mice with tMCAO revealed that bone marrow cell cycling peaked 4 days after stroke (P<0.05 versus pre tMCAO). Flow cytometry and cell cycle analysis showed activation of the entire hematopoietic tree, including myeloid progenitors. The cycling fraction of the most upstream hematopoietic stem cells increased from 3.34%±0.19% to 7.32%±0.52% after tMCAO (P<0.05). In vivo microscopy corroborated proliferation of adoptively transferred hematopoietic progenitors in the bone marrow of mice with stroke. The hematopoietic system’s myeloid bias was reflected by increased expression of myeloid transcription factors, including PU.1 (P<0.05), and by a decline in lymphocyte precursors. In mice after tMCAO, tyrosine hydroxylase levels in sympathetic fibers and bone marrow noradrenaline levels rose (P<0.05, respectively), associated with a decrease of hematopoietic niche factors that promote stem cell quiescence. In mice with genetic deficiency of the &bgr;3 adrenergic receptor, hematopoietic stem cells did not enter the cell cycle in increased numbers after tMCAO (naive control, 3.23±0.22; tMCAO, 3.74±0.33, P=0.51). Conclusions: Ischemic stroke activates hematopoietic stem cells via increased sympathetic tone, leading to a myeloid bias of hematopoiesis and higher bone marrow output of inflammatory Ly6Chigh monocytes and neutrophils.

FGF/FGFR signaling in bone formation: Progress and perspectives

Fibroblast growth factors (FGFs) are important molecules that control bone formation. FGF act by activating FGF receptors (FGFRs) and downstream signaling pathways that control cells of the osteoblast lineage. Recent advances have been made in the identification of FGF/FGFR signaling pathways that control osteogenesis. Indeed, studies of mouse and human models provided novel insights into the signaling pathways that control bone formation. Genomic studies also highlighted the implication of molecular targets of FGF/FGFR signaling regulating osteoblastogenesis. Recent studies further revealed the important role of crosstalks between FGF/FGFR signaling and other signaling pathways in the regulation of osteogenesis. Finally, the importance of the mechanisms modulating FGFR degradation in the control of osteoblast differentiation has been recently revealed. This short review summarizes the recently described mechanisms underlying FGF/FGFR signaling that are involved in the control of osteoblastogenesis. This knowledge may have potential therapeutic implications in skeletal disorders characterized by abnormal bone formation.

E3 ubiquitin ligase-mediated regulation of bone formation and tumorigenesis.

The ubiquitination–proteasome and degradation system is an essential process that regulates protein homeostasis. This system is involved in the regulation of cell proliferation, differentiation and survival, and dysregulations in this system lead to pathologies including cancers. The ubiquitination system is an enzymatic cascade that mediates the marking of target proteins by an ubiquitin label and thereby directs their degradation through the proteasome pathway. The ubiquitination of proteins occurs through a three-step process involving ubiquitin activation by the E1 enzyme, allowing for the transfer to a ubiquitin-conjugated enzyme E2 and to the targeted protein via ubiquitin-protein ligases (E3), the most abundant group of enzymes involved in ubiquitination. Significant advances have been made in our understanding of the role of E3 ubiquitin ligases in the control of bone turnover and tumorigenesis. These ligases are implicated in the regulation of bone cells through the degradation of receptor tyrosine kinases, signaling molecules and transcription factors. Initial studies showed that the E3 ubiquitin ligase c-Cbl, a multi-domain scaffold protein, regulates bone resorption by interacting with several molecules in osteoclasts. Further studies showed that c-Cbl controls the ubiquitination of signaling molecules in osteoblasts and in turn regulates osteoblast proliferation, differentiation and survival. Recent data indicate that c-Cbl expression is decreased in primary bone tumors, resulting in excessive receptor tyrosine kinase signaling. Consistently, c-Cbl ectopic expression reduces bone tumorigenesis by promoting tyrosine kinase receptor degradation. Here, we review the mechanisms of action of E3 ubiquitin ligases in the regulation of normal and pathologic bone formation, and we discuss how targeting the interactions of c-Cbl with some substrates may be a potential therapeutic strategy to promote osteogenesis and to reduce tumorigenesis.

Molecular silencing of Twist1 enhances osteogenic differentiation of murine mesenchymal stem cells: Implication of FGFR2 signaling

The capacity of mesenchymal stem cells (MSCs) to differentiate into functional osteoblasts is tightly controlled by transcription factors that trigger osteoblast commitment and differentiation. The role of Twist1, a basic helix‐loop‐helix (bHLH) transcription factor, in osteogenic differentiation of MSCs remains unclear. Here we investigated the role of Twist1 in the osteogenic differentiation program of murine C3H10T1/2 mesenchymal cells. We showed that molecular silencing of Twist1 using short hairpin RNA (shRNA) expression moderately increased C3H10T1/2 cell proliferation and had no effect on cell survival. In contrast, Twist1 silencing enhanced osteoblast gene expression and matrix mineralization in vitro. Biochemical analyses revealed that Twist1 silencing increased the expression of FGFR2 protein level, which was reduced by a mutant Runx2. Consistent with this finding, Twist1 silencing increased ERK1/2 and PI3K signaling. Moreover, molecular or pharmacological inhibition of FGFR2 or of ERK1/2 and PI3K signaling partly abolished the increased osteoblast gene expression induced by Twist1 silencing in C3H10T1/2 cells. These results reveal that Twist1 silencing upregulates osteoblast differentiation of murine mesenchymal cells in part via activation of FGFR2 expression and downstream signaling pathways, which provides novel insights into the molecular signals by which this transcription factor regulates the osteogenic differentiation program in MSCs. J. Cell. Biochem. 110: 1147–1154, 2010. Published 2010 Wiley‐Liss, Inc.

The Casitas B Lineage Lymphoma (Cbl) Mutant G306E Enhances Osteogenic Differentiation in Human Mesenchymal Stromal Cells in Part by Decreased Cbl-mediated Platelet-derived Growth Factor Receptor α and Fibroblast Growth Factor Receptor 2 Ubiquitination

Human bone marrow-derived mesenchymal stromal cells (hMSCs) have the capacity to differentiate into several cell types including osteoblasts and are therefore an important cell source for bone tissue regeneration. A crucial issue is to identify mechanisms that trigger hMSC osteoblast differentiation to promote osteogenic potential. Casitas B lineage lymphoma (Cbl) is an E3 ubiquitin ligase that ubiquitinates and targets several molecules for degradation. We hypothesized that attenuation of Cbl-mediated degradation of receptor tyrosine kinases (RTKs) may promote osteogenic differentiation in hMSCs. We show here that specific inhibition of Cbl interaction with RTKs using a Cbl mutant (G306E) promotes expression of osteoblast markers (Runx2, alkaline phosphatase, type 1 collagen, osteocalcin) and increases osteogenic differentiation in clonal bone marrow-derived hMSCs and primary hMSCs. Analysis of molecular mechanisms revealed that the Cbl mutant increased PDGF receptor α and FGF receptor 2 but not EGF receptor expression in hMSCs, resulting in increased ERK1/2 and PI3K signaling. Pharmacological inhibition of FGFR or PDGFR abrogated in vitro osteogenesis induced by the Cbl mutant. The data reveal that specific inhibition of Cbl interaction with RTKs promotes the osteogenic differentiation program in hMSCs in part by decreased Cbl-mediated PDGFRα and FGFR2 ubiquitination, providing a novel mechanistic approach targeting Cbl to promote the osteogenic capacity of hMSCs.

Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils

A bona fide portrayal of tumor growth Bone has a well-established role in advanced cancer. It provides a supportive microenvironment for the growth of metastatic cells that escape the primary tumor, which ultimately leads to loss of bone mass. Engblom et al. show that bone may also contribute to early-stage tumorigenesis through a mechanism that leads to an increase in bone mass (see the Perspective by Zhang and Lyden). In mouse models of lung adenocarcinoma, primary tumor cells remotely activated bone-resident cells called osteoblasts, which have a bone-building function. The activated osteoblasts in turn triggered production of a certain type of neutrophil that infiltrates the primary tumor and promotes its growth. Patients with early-stage lung cancer were also found to have an increase in bone density, consistent with the findings in mice. Science, this issue p. eaal5081; see also p. 1127 Systemic cross-talk between tumor and bone can boost the growth of early-stage lung cancer in mice. INTRODUCTION Myeloid cells have emerged as key regulators of cancer growth because of their abundance in the tumor stroma in a broad range of cancers, their association with clinical outcome, and their ability to modulate tumor progression. Most tumor-infiltrating myeloid cells derive from circulating precursors, which are produced in distant tissues, and some tumors amplify myeloid cell activity by skewing hematopoiesis toward the myeloid lineage or increasing myeloid cell populations in the periphery. For example, patients across diverse cancer types can present with elevated levels of myeloid progenitor cells in peripheral blood. Additionally, increased numbers of circulating myeloid cells, such as neutrophils, often correlate with poorer clinical outcome. It is therefore important to consider host changes that occur away from the tumor stroma to more fully understand the biological processes underlying tumor growth. RATIONALE The bone marrow is a tissue of particular interest as it is the main production site for hematopoietic cells corresponding to all circulating blood lineages in the adult. The marrow contains resident cell components, such as osteoblasts, which not only participate in bone maintenance but also regulate hematopoiesis and immune cell fate. However, our understanding of bone dynamics in the context of cancer (growing at sites distant from the local bone microenvironment) and related immune responses remains limited. To address this knowledge gap, we explored whether a common solid cancer—lung adenocarcinoma—remotely affects bone tissue and how this might shape tumor-associated hematopoietic responses and tumor growth. RESULTS We show in different mouse models and in cancer patients (n = 70) that lung adenocarcinomas increase bone stromal activity even in the absence of local metastasis. Animal studies further reveal that the cancer-induced bone phenotype involves bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. Ocn+ cells affect distant tumor progression because experimentally reducing the number of these cells limits lung tumor growth. Also, Ocn+ cells are required for full-fledged tumor infiltration by a distinct subset of neutrophils that are defined by their high expression of the lectin SiglecF (sialic acid–binding immunoglobulin-like lectin F). Compared to other neutrophils, SiglecFhigh cells express genes associated with cancer-promoting processes, including angiogenesis, myeloid cell differentiation and recruitment, extracellular matrix remodeling, suppression of T cell responses, and tumor cell proliferation and growth. Additionally, SiglecFhigh neutrophils have increased reactive oxygen species production, promote macrophage differentiation, and boost tumor progression in vivo. We further report that the soluble receptor for advanced glycation end products (sRAGE) is up-regulated in the circulation of tumor-bearing mice and fosters osteoblastic activity and osteoblast-dependent neutrophil maturation in vitro. CONCLUSION This study identifies systemic cross-talk between lung tumors and bones: Lung tumors can remotely activate Ocn+ osteoblastic cells in bones even in the absence of local metastasis. In turn, these Ocn+ cells supply tumors with SiglecFhigh neutrophils, which foster cancer progression. The findings bear scientific and therapeutic importance because they reveal contributions of the host systemic environment to tumor growth and they position Ocn+ cells, SiglecFhigh neutrophils, and sRAGE as candidate clinical biomarkers and possible intervention points for anticancer therapy. Systemic cross-talk between lung tumors and bones. Lung adenocarcinomas can remotely activate Ocn+ osteoblastic cells in bones even in the absence of local metastasis. In turn, these osteoblasts supply tumors with SiglecFhigh neutrophils, which exhibit cancer-promoting functions (left). By contrast, the bone marrow in steady state only produces SiglecFlow neutrophils (right). Bone marrow–derived myeloid cells can accumulate within tumors and foster cancer outgrowth. Local immune-neoplastic interactions have been intensively investigated, but the contribution of the systemic host environment to tumor growth remains poorly understood. Here, we show in mice and cancer patients (n = 70) that lung adenocarcinomas increase bone stromal activity in the absence of bone metastasis. Animal studies reveal that the cancer-induced bone phenotype involves bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh neutrophils, which exhibit cancer-promoting properties. Experimentally reducing Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth. These observations posit osteoblasts as remote regulators of lung cancer and identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven protumoral response.

Promotion of Osteoblast Differentiation in Mesenchymal Cells Through Cbl‐Mediated Control of STAT5 Activity

The identification of the molecular mechanisms controlling the degradation of regulatory proteins in mesenchymal stromal cells (MSC) may provide clues to promote MSC osteogenic differentiation and bone regeneration. Ubiquitin ligase‐dependent degradation of proteins is an important process governing cell fate. In this study, we investigated the role of the E3 ubiquitin ligase c‐Cbl in MSC osteoblast differentiation and identified the mechanisms involved in this effect. Using distinct shRNA targeting c‐Cbl, we showed that c‐Cbl silencing promotes osteoblast differentiation in murine and human MSC, as demonstrated by increased alkaline phosphatase activity, expression of phenotypic osteoblast marker genes (RUNX2, ALP, type 1 collagen), and matrix mineralization in vitro. Coimmunoprecipitation analyses showed that c‐Cbl interacts with the transcription factor STAT5, and that STAT5 forms a complex with RUNX2, a master transcription factor controlling osteoblastogenesis. Silencing c‐Cbl decreased c‐Cbl‐mediated STAT5 ubiquitination, increased STAT5 protein level and phosphorylation, and enhanced STAT5 and RUNX2 transcriptional activity. The expression of insulin like growth factor‐1 (IGF‐1), a target gene of STAT5, was increased by c‐Cbl silencing in MSC and in bone marrow stromal cells isolated from c‐Cbl deficient mice, suggesting that IGF‐1 contributes to osteoblast differentiation induced by c‐Cbl silencing in MSC. Consistent with these findings, pharmacological inhibition of STAT5 activity, or neutralization of IGF‐1 activity, abrogated the positive effect of c‐Cbl knockdown on MSC osteogenic differentiation. Taken together, the data provide a novel functional mechanism by which the ubiquitin ligase c‐Cbl regulates the osteoblastic differentiation program in mesenchymal cells by controlling Cbl‐mediated STAT5 degradation and activity. STEM Cells2013;31:1340–1349

Targeting the E3 Ubiquitin Casitas B-Lineage Lymphoma Decreases Osteosarcoma Cell Growth and Survival and Reduces Tumorigenesis

Targeting receptor tyrosine kinase (RTK) degradation may be an interesting approach to reduce RTK cell signaling in cancer cells. Here we show that increasing E3 ubiquitin ligase casitas B‐lineage lymphoma (c‐Cbl) expression using lentiviral infection decreased osteosarcoma cell replication and survival and reduced cell migration and invasion in murine and human osteosarcoma cells. Conversely, c‐Cbl inhibition using short hairpin RNA (shRNA) increased osteosarcoma cell growth and survival, as well as invasion and migration, indicating that c‐Cbl plays a critical role as a bone tumor suppressor. Importantly, the anticancer effect of increasing c‐Cbl expression in osteosarcoma cells was related mainly to the downregulation of epidermal growth factor receptor (EGFR) and platelet‐derived growth factor receptor alpha (PDGFRα). In a murine bone tumor model, increasing c‐Cbl expression also reduced RTK expression, resulting in decreased tumor cell proliferation and survival and reduced tumor growth. Interestingly, increasing c‐Cbl also markedly reduced lung metastasis in mice. Tissue microarray analysis revealed that low c‐Cbl protein expression is associated with elevated EGFR and PDGFRα protein levels in human osteosarcoma with poor outcome. This study shows that increasing c‐Cbl expression reduces osteosarcoma cell growth, survival, and metastasis in part through downregulation of RTKs, which supports the potential therapeutic interest of targeting c‐Cbl in malignant bone diseases involving increased RTK.

ErbB3 silencing reduces osteosarcoma cell proliferation and tumor growth in vivo

Osteosarcoma is the most common primary bone tumor in children and adults. Despite improved prognosis, resistance to chemotherapy remains responsible for failure of osteosarcoma treatment. The identification of the molecular signals that contribute to the aberrant osteosarcoma cell growth may provide clues to develop new therapeutic strategies for chemoresistant osteosarcoma. Here we show that the expression of ErbB3 is increased in human osteosarcoma cells in vitro. Tissue microarray analysis of tissue cores from osteosarcoma patients further showed that the ErbB3 protein expression is higher in bone tumors compared to normal bone tissue, and is further increased in patients with recurrent disease or soft tissue metastasis. In murine osteosarcoma cells, silencing ErbB3 using shRNA decreased cell replication, cell migration and invasion, indicating that ErbB3 contributes to tumor cell growth and invasiveness. Furthermore, ErbB3 silencing markedly reduced tumor growth in a murine allograft model in vivo. Immunohistochemal analysis showed that the reduced tumor growth induced by ErbB3 silencing in this model resulted from decreased cell osteosarcoma cell proliferation, supporting a role of ErbB3 in bone tumor growth in vivo. Taken together, the results reveal that ErbB3 expression in human osteosarcoma correlates with tumor grade. Furthermore, silencing ErbB3 in a murine osteosarcoma model results in decreased cell growth and invasiveness in vitro, and reduced tumor growth in vivo, which supports the potential therapeutic interest of targeting ErbB3 in osteosarcoma.

Hematopoiesis: Reconciling Historic Controversies about the Niche

The niche, as first conceptualized, was in conflict with the prevailing wisdom that stem cells have internal logic. The niche hypothesis has been indisputably confirmed. Yet, recent findings indicate little plasticity of epigenetically scripted hematopoietic stem/progenitors. Reconciling this conflict requires re-envisioning the niche as an enabler, not designer, of cell fate.